Nitric oxide-releasing imidate and thioimidate diazeniumdiolates

ABSTRACT

The invention provides NO- or NO − -releasing imidate; and thioimidate, diazeniumdiolates, in which the N 2 O 2   −  functional group is bonded to a carbon atom. The imidate and thioimidate diazeniumdiolates are bound to a polymer or a substrate.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This patent application is a divisional of U.S. patent application Ser.No. 09/950,162, filed Sep. 10, 2001, now U.S. Pat. No. 6,673,338.

FIELD OF THE INVENTION

This invention relates to nitric oxide-releasing imidate and thioimidatediazeniumdiolates, to compositions comprising such compounds, to methodsof using such compounds and compositions, and to a method for thepreparation of nitric oxide-releasing imidate and thioimidatediazeniumdiolates.

BACKGROUND OF THE INVENTION

Nitric oxide (NO) has been implicated as part of a cascade ofinteracting agents involved in a wide variety of bioregulatoryprocesses, including the physiological control of blood pressure,macrophage-induced cytostasis and cytotoxicity, and neurotransmission(Moncada et al., “Nitric Oxide from L-Arginine: A Bioregulatory System,”Excerpta Medica, International Congress Series 897, Elsevier SciencePublishers B.V.: Amsterdam (1990); Marletta et al., Biofactors 2:219–225 (1990); Ignarro, Hypertension (Dallas) 16: 477–483 (1990);Kerwin et al., J. Med. Chem. 38: 4343–4362 (1995); and Anggar{acute over(d)}, Lancet 343: 1199–1206 (1994)). Given that NO plays a role in sucha wide variety of bioregulatory processes, great effort has beenexpended to develop compounds capable of releasing NO. Some of thesecompounds are capable of releasing NO spontaneously, e.g., by hydrolysisin aqueous media, whereas others are capable of releasing NO upon beingmetabolized (Lefer et al., Drugs Future 19: 665–672 (1994)).

Several types of compounds of the general structure

have been known for many years. Traube (Liebigs Ann. Chem. 300: 81–123(1898)) reported the preparation of a number of such compounds and notedthat treatment of the compounds with acid produced a “brown gas.” Giventhat the brown gas is nitrogen dioxide which may be produced directly,the release of brown gas by the compounds prepared by Traube is not, inand of itself, evidence of NO release. Compounds of the structural typereported by Traube are known to require harsh treatment with mineralacids to release any gas and such treatment is, of course, incompatiblewith biological utility.

Another compound, which has the structure

and which has been named cupferron, has been shown by Kubrina et al.,(Izvestia Akademii Nauk SSSR Seriia Biologicheskaia 6: 844–850 (1988)English Trans.: Biol. Bull. Acad. Sci. USSR. 533–538, (1988)) togenerate NO in vivo. In addition, the antibiotics alanosine(C(O)(OH)CH(NH₂)CH₂ N(O)═NOH) and dopastin (CH₃CH═CHC(O)NHCH₂CH(i-propyl)-N(O)═NOH), as well as cupferron, have been shown to releaseNO in vivo by enzymatic oxidation (Alston et al., J. Biol. Chem. 260:4069–4074 (1985)).

Many of the known diazeniumdiolates and their clinical applications aredisclosed in recently issued patents. For example, U.S. Pat. No.4,954,526 (Keefer et al.) discloses nitric oxide-primary amine complexesuseful as cardiovascular agents. U.S. Pat. No. 5,039,705 (Keefer et al.)and U.S. Pat. No. 5,208,233 (Keefer et al.) disclose anti-hypertensivecompositions of secondary amine-nitric oxide adducts. U.S. Pat. No.5,155,137 (Keefer et al.) and U.S. Pat. No. 5,250,550 (Keefer et al.)disclose complexes of nitric oxide with polyamines. U.S. Pat. No.5,405,919 (Keefer et al.), U.S. Pat. No. 5,525,357 (Keefer et al.) andU.S. Pat. No. 5,718,892 (Keefer et al.) disclose polymer-bound nitricoxide/nucleophile adduct compositions. U.S. Pat. No. 5,366,997 (Keeferet al.) discloses oxygen-substituted derivatives of nucleophile-nitricoxide adducts as nitric oxide donor drugs. U.S. Pat. No. 5,389,675(Christodoulou et al.) discloses mixed ligand complexes of nitricoxide-nucleophile adducts. U.S. Pat. No. 5,632,981 (Saavedra et al.)discloses biopolymer-bound nitric oxide-releasing compositions. U.S.Pat. No. 5,691,423 (Smith et al.) discloses polysaccharide-bound nitricoxide-nucleophile adducts. U.S. Pat. No. 5,721,365 (Keefer et al.)discloses N-substituted piperazine diazeniumdiolates. U.S. Pat. No.5,185,376 (Diodati et al.) discloses therapeutic inhibition of plateletaggregation by nucleophile-nitric oxide complexes. U.S. Pat. No.5,650,447 (Keefer et al.) discloses nitric oxide-releasing polymers totreat restenosis and related disorders. U.S. Pat. No. 5,676,963 (Keeferet al.) discloses implants, prostheses, and stents comprisingpolymer-bound nitric oxide/nucleophile adducts capable of releasingnitric oxide. U.S. Pat. No. 5,700,830 (Korthius et al.) discloses theuse of nitric oxide adducts for reducing metastatic risk. U.S. Pat. No.5,714,511 (Saavedra et al.) U.S. Pat. No. 5,814,666 (Keefer et al.)disclose selective prevention of organ injury in sepsis and shock usingselective release of nitric oxide in vulnerable organs. U.S. Pat. No.5,731,305 (Keefer et al.) discloses anti-hypertension compositions ofsecondary amine-nitric oxide adducts. U.S. Pat. No. 5,910,316 (Keefer etal.) discloses encapsulated and non-encapsulated nitric oxide generatorsuseful as antimicrobial agents.

Other diazeniumdiolates useful in a host of applications include thosedescribed in U.S. patent application Ser. No. 09/254,301 (Saavedra etal.), which discloses O²-arylated and O²-glycosylated diazeniumdiolates,and U.S. Pat. No. 6,232,336 (Hrabie et al.), which discloses amidine- orenamine-derived diazeniumdiolates.

Despite the extensive literature available on NO and nitricoxide-releasing compounds, there remains a need for stable nitricoxide-releasing compounds in which the nitric oxide-releasing group N₂O₂⁻ is bonded directly to a carbon atom and that serve as versatileintermediates in the preparation of a wide variety of therapeutic nitricoxide-releasing compounds.

BRIEF SUMMARY OF THE INVENTION

The invention described herein provides for a novel class of imidate andthioimidate diazeniumdiolates and their preparation. The imidate andthioimidate diazeniumdiolates are useful therapeutic compounds and serveas important building blocks in the synthesis of other biologicallyimportant NO-releasing compounds. These and other advantages of thepresent invention, as well as additional inventive features, will beapparent from the description of the invention provided below.

The invention provides NO- or NO⁻-releasing imidate or thioimidatediazeniumdiolates in which the N₂O₂ ⁻ functional group is bonded to acarbon atom. The invention also provides compositions comprising suchdiazeniumdiolate compounds, and methods of using such compounds andcompositions. The invention further provides a method for thepreparation of NO- or NO⁻-releasing imidate or thioimidatediazeniumdiolates.

DETAILED DESCRIPTION OF THE INVENTION

The novel class of imidate and thioimidate diazeniumdiolates is capableof releasing nitric oxide under physiological conditions. Thesecompounds are useful for treating biological conditions where a releaseof nitric oxide is beneficial.

The invention provides a novel class of NO-releasing imidate andthioimidate diazeniumdiolates of the formula:

wherein X is O or S; R¹ is hydrogen, an unsubstituted or substitutedC₁₋₁₂ straight chain alkyl, an unsubstituted or substituted C₃₋₁₂branched chain alkyl, an unsubstituted or substituted C₃₋₁₂ straightchain alkenyl, an unsubstituted or substituted C₃₋₁₂ branched chainalkenyl, an unsubstituted or substituted alkoxy, nitrile, halo, anunsubstituted or substituted benzyl, an unsubstituted or substitutedphenyl, an unsubstituted or substituted naphthyl, an unsubstituted orsubstituted piperazino, an unsubstituted or substituted morpholino,amino, an unsubstituted or substituted alkylamino, an unsubstituted orsubstituted arylamino, an unsubstituted or substituted dialkylamino, anunsubstituted or substituted diarylamino, carboxyalkylamino,carboxydialkylamino, an unsubstituted or substituted tolyl, xylyl,anisyl, mesityl, an unsubstituted or substituted acetoxy, carboxy, anunsubstituted or substituted carboxyethyl, an unsubstituted orsubstituted alkylcarbonyl, thiol, an unsubstituted or substitutedalkylthio, an unsubstituted or substituted alkyloxy, carboxamido, anunsubstituted or substituted alkylcarboxamido, an unsubstituted orsubstituted dialkylcarboxamido, an unsubstituted or substituted phenoxy,an unsubstituted or substituted benzyloxy, phenylcarbonyl,benzylcarbonyl, an unsubstituted or substituted nitrophenyl,trialkylsilyl or nitro; R² is an unsubstituted or substituted C₁₋₁₂straight chain alkyl or an unsubstituted,substituted C₃₋₁₂ branchedchain alkyl, a phenyl, or naphthyl; R³ is hydrogen, an unsubstituted orsubstituted C₁₋₁₂ straight chain alkyl, an unsubstituted or substitutedC₃₋₁₂ branched chain alkyl, an unsubstituted or substituted C₃₋₁₂straight chain alkenyl, an unsubstituted or substituted C₃₋₁₂ branchedchain alkenyl, an unsubstituted or substituted C₃₋₈ cycloalkyl, a C₃₋₈heterocyclic ring, an unsubstituted or substituted naphthyl, anunsubstituted or substituted tetrahydronaphthyl, an unsubstituted orsubstituted octahydronaphthyl, benzyl or substituted benzyl, or phenylor substituted phenyl; and M^(+x) is a pharmaceutically acceptablecation, x is the valence of the cation, and a and b are the smallestintegers that result in a neutral compound. The substituent R¹ can befurther substituted with at least one nitric oxide-releasing functionalgroup.

Any one or more of R¹, R², and R³ of formula (I) can be substituted.Generally each of R¹, R², and R³ can have 1 to 3 substituents that areindependently selected from the group consisting of alkyl, aryl, such asphenyl or naphthyl, alkoxy, aryloxy, acyloxy, benzyl, benzyloxy, acetyl,carboxyl, carboxyalkyl, carboxyalkylamido, carboxydialkylamido,carboxamido, alkylcarbonyl, arylamino, diarylamino, nitrile, tolyl,xylyl, mesityl, anisyl, pyrrolidinyl, formyl, dioxane, alkylthiol,heteroaryl, such as pyran, pyrrole, furan, thiophene, thiazole,pyrazole, pyridine, or pyrimidine, phenylcarbonyl, benzylcarbonyl,nitrophenyl, trialkylsilyl, nitro, sulfonyl, nitrobenzyl,trialkylammonium, tetrahydrofuranyl, tetrahydropyranyl, piperdinyl,morpholinyl, halo, cyano, hydroxy, thiol, cycloalkyl, amino, alkylamino,and dialkylamino.

In one aspect, the present invention provides novel nitricoxide-releasing imidate compounds of the formula (II):

wherein R¹ is hydrogen, an unsubstituted or substituted C₁₋₁₂ straightchain alkyl, an unsubstituted or substituted C₃₋₁₂ branched chain alkyl,an unsubstituted or substituted C₃₋₁₂ straight chain alkenyl, anunsubstituted or substituted C₃₋₁₂ branched chain alkenyl, anunsubstituted or substituted alkoxy, nitrile, halo, an unsubstituted orsubstituted benzyl, an unsubstituted or substituted phenyl, anunsubstituted or substituted naphthyl, an unsubstituted or substitutedpiperazino, an unsubstituted or substituted morpholino, amino, anunsubstituted or substituted alkylamino, an unsubstituted or substitutedarylamino, an unsubstituted or substituted dialkylamino, anunsubstituted or substituted diarylamino, carboxyalkylamino,carboxydialkylamino, an unsubstituted or substituted tolyl, xylyl,anisyl, mesityl, an unsubstituted or substituted acetoxy, carboxy, anunsubstituted or substituted carboxyethyl, an unsubstituted orsubstituted alkylcarbonyl, thiol, an unsubstituted or substitutedalkylthio, an unsubstituted or substituted alkyloxy, carboxamido, anunsubstituted or substituted alkylcarboxamido, an unsubstituted orsubstituted dialkylcarboxamido, an unsubstituted or substituted phenoxy,an unsubstituted or substituted benzyloxy, phenylcarbonyl,benzylcarbonyl, an unsubstituted or substituted nitrophenyl,trialkylsilyl or nitro; R² is an unsubstituted or substituted C₁₋₁₂straight chain alkyl, an unsubstituted or substituted C₃₋₁₂ branchedchain alkyl, a phenyl, or naphthyl; R³ is hydrogen, an unsubstituted orsubstituted C₁₋₁₂ straight chain alkyl, an unsubstituted or substitutedC₃₋₁₂ branched chain alkyl, an unsubstituted or substituted C₃₋₁₂straight chain alkenyl, an unsubstituted or substituted C₃₋₁₂ branchedchain alkenyl, an unsubstituted or substituted C₃₋₈ cycloalkyl, a C₃₋₈heterocyclic ring, an unsubstituted or substituted naphthyl, anunsubstituted or substituted tetrahydronaphthyl, an unsubstituted orsubstituted octahydronaphthyl, benzyl or substituted benzyl, or phenylor substituted phenyl; and M^(+x) is a pharmaceutically acceptablecation, x is the valence of the cation, and a and b are the smallestintegers that result in a neutral compound. The substituent R¹ can befurther substituted with at least one nitric oxide-releasing functionalgroup.

Any one or more of R¹, R², and R³ of formula (II) can be substituted.Generally each of R¹, R², and R³ can have 1 to 3 substituents that areindependently selected from the group consisting of alkyl, aryl, such asphenyl or naphthyl, alkoxy, aryloxy, acyloxy, benzyl, benzyloxy, acetyl,carboxyl, carboxyalkyl, carboxyalkylamido, carboxydialkylamido,carboxamido, alkylcarbonyl, arylamino, diarylamino, nitrile, tolyl,xylyl, mesityl, anisyl, pyrrolidinyl, formyl, dioxane, alkylthiol,heteroaryl, such as pyran, pyrrole, furan, thiophene, thiazole,pyrazole, pyridine, or pyrimidine, phenylcarbonyl, benzylcarbonyl,nitrophenyl, trialkylsilyl, nitro, sulfonyl, nitrobenzyl,trialkylammonium, tetrahydrofuranyl, tetrahydropyranyl, piperdinyl,morpholinyl, halo, cyano, hydroxy, thiol, cycloalkyl, amino, alkylamino,and dialkylamino.

Preferably, R¹ of the compound of formula (II) is an unsubstituted orsubstituted aryl. More preferably, R¹ of the compound of formula (II) isan unsubstituted or substituted phenyl, and most preferably R¹ of thecompound of formula (II) is a phenyl, 4-methoxyphenyl, 4-chlorophenyl,or 3,4,5-trimethoxyphenyl. Preferably, R² of the compound of formula(II) is methyl or ethyl. More preferably, R² of the compound of formula(II) is methyl. Preferably, R³ of the compound of formula (II) ishydrogen. More preferably, R¹ of the compound of formula (II) is anunsubstituted aryl, R² is methyl, and R³ is hydrogen.

In another aspect, the present invention provides novel nitricoxide-releasing thioimidate compounds of formula (III):

wherein R¹ is hydrogen, an unsubstituted or substituted C₁₋₁₂ straightchain alkyl, an unsubstituted or substituted C₃₋₁₂ branched chain alkyl,an unsubstituted or substituted C₃₋₁₂ straight chain alkenyl, anunsubstituted or substituted C₃₋₁₂ branched chain alkenyl, anunsubstituted or substituted alkoxy, nitrile, halo, an unsubstituted orsubstituted benzyl, an unsubstituted or substituted phenyl, anunsubstituted or substituted naphthyl, an unsubstituted or substitutedpiperazino, an unsubstituted or substituted morpholino, amino, anunsubstituted or substituted alkylamino, an unsubstituted or substitutedarylamino, an unsubstituted or substituted dialkylamino, anunsubstituted or substituted diarylamino, carboxyalkylamino,carboxydialkylamino, an unsubstituted or substituted tolyl, xylyl,anisyl, mesityl, an unsubstituted or substituted acetoxy, carboxy, anunsubstituted or substituted carboxyethyl, an unsubstituted orsubstituted alkylcarbonyl, thiol, an unsubstituted or substitutedalkylthio, an unsubstituted or substituted alkyloxy, carboxamido, anunsubstituted or substituted alkylcarboxamido, an unsubstituted orsubstituted dialkylcarboxamido, an unsubstituted or substituted phenoxy,an unsubstituted or substituted benzyloxy, phenylcarbonyl,benzylcarbonyl, an unsubstituted or substituted nitrophenyl,trialkylsilyl or nitro; R² is an unsubstituted or substituted C₁₋₁₂straight chain alkyl, an unsubstituted or substituted C₃₋₁₂ branchedchain alkyl, a phenyl, or naphthyl; R³ is hydrogen, an unsubstituted orsubstituted C₁₋₁₂ straight chain alkyl, an unsubstituted or substitutedC₃₋₁₂ branched chain alkyl, an unsubstituted or substituted C₃₋₁₂straight chain alkenyl, an unsubstituted or substituted C₃₋₁₂ branchedchain alkenyl, an unsubstituted or substituted C₃₋₈ cycloalkyl, a C₃₋₈heterocyclic ring, an unsubstituted or substituted naphthyl, anunsubstituted or substituted tetrahydronaphthyl, an unsubstituted orsubstituted octahydronaphthyl, benzyl or substituted benzyl, or phenylor substituted phenyl; and M^(+x) is a pharmaceutically acceptablecation, x is the valence of the cation, and a and b are the smallestintegers that result in a neutral compound. The substituent R¹ can befurther substituted with at least one nitric oxide-releasing functionalgroup.

Any one or more of R¹, R², and R³ of formula (III) can be substituted.Generally each of R¹, R², and R³ can have 1 to 3 substituents that areindependently selected from the group consisting of alkyl, aryl, such asphenyl or naphthyl, alkoxy, aryloxy, acyloxy, benzyl, benzyloxy, acetyl,carboxyl, carboxyalkyl, carboxyalkylamido, carboxydialkylamido,carboxamido, alkylcarbonyl, arylamino, diarylamino, nitrile, tolyl,xylyl, mesityl, anisyl, pyrrolidinyl, formyl, dioxane, alkylthiol,heteroaryl, such as pyran, pyrrole, furan, thiophene, thiazole,pyrazole, pyridine, or pyrimidine, phenylcarbonyl, benzylcarbonyl,nitrophenyl, trialkylsilyl, nitro, sulfonyl, nitrobenzyl,trialkylammonium, tetrahydrofuranyl, tetrahydropyranyl, piperdinyl,morpholinyl, halo, cyano, hydroxy, thiol, cycloalkyl, amino, alkylamino,and dialkylamino.

Preferably, R¹ of the compound of formula (III) is an unsubstituted orsubstituted aryl. More preferably, R¹ of the compound of formula (III)is an unsubstituted or substituted phenyl, and most preferably R¹ of thecompound of formula (III) is a phenyl, 4-methoxyphenyl, 4-chlorophenyl,or 3,4,5-trimethoxyphenyl. Preferably, R² of the compound of formula(III) is methyl or ethyl. More preferably, R² of the compound of formula(III) is methyl. Preferably, R³ of the compound of formula (III) ishydrogen. More preferably, R¹ of the compound of formula (III) is anunsubstituted aryl, R² is methyl, and R³ is hydrogen.

The counterion, M^(+x), is any pharmaceutically acceptable counterion.The only requirement for the pharmaceutically acceptable counterionchosen is biological compatability in an animal, such as a human.Biologically acceptable counterions include alkali metals such as sodiumion, potassium ion, lithium ion, and the like; alkaline earth metalssuch as magnesium ion, calcium ion, and the like; Group III metals suchas aluminum ion; Group IV metals such as tin ion; and transition metals,including iron ion, copper ion, manganese ion, zinc ion, cobalt ion,vanadium ion, molybdenum ion, platinum ion, and the like. Non-metalcounterions include quaternary ammonium ions. Metal ions that may beconsidered toxic may, nevertheless, be pharmaceutically acceptable andthus within the scope of the invention if their complexes with thediazeniumdiolates are sufficiently potent pharmacologically and thetotal concentration of the metal counterion upon dosing is below thetoxic threshold of the metal.

In another aspect, the invention provides a method of preparing a nitricoxide-releasing compound of formula (I) from a compound not containingan imidate or thioimidate group, as illustrated in Equation 1. In thepreparation of an imidate diazeniumdiolate, the method comprises (a)contacting a nitrile of the formula R¹CH₂CN with a metal alkoxide and analcohol (e.g., NaOMe/HOMe); and (b) contacting the product of (a) withnitric oxide to form a diazeniumdiolated nitrile compound; (c)contacting the diazeniumdiolated nitrile compound with an alkoxide toform the imidate of formula (II), for example, where R¹ is discussedherein. Similarly, in the preparation of a thioimidate diazeniumdiolate,the method comprises (a) contacting a nitrile of the formula R¹CH₂CNwith a metal thiolate and a thiol (e.g., NaSMe/HSMe); and (b) contactingthe product of (a) with nitric oxide to form a diazeniumdiolated nitrilecompound; (c) contacting the diazeniumdiolated nitrile compound with athiolate to form the thioimidate of formula (III), wherein R¹ isdiscussed herein. These methods in accordance with the invention areuseful for preparing a (thio)imidate diazeniumdiolate in which theNO-releasing N₂O₂ ⁻ functional groups are bound to a carbon atom ratherthan nitrogen.

wherein X is O or S.

While typically the reaction to form the diazeniumdiolated imidatesproceeds as described above, it is recognized that in some casesformation of the imidate may precede attachment of the diazeniumdiolategroup. The exact sequence of the reaction is of no consequence to theultimate goals of the invention and should in no way be construed tolimit its scope.

While not wishing to be bound by any particular theory, it is believedthat the nitrile R¹CH₂CN, in the presence of alkoxide or thiolate, formsa carbanion. This carbanion forms a bis-diazeniumdiolate compound in thepresence of nitric oxide gas. Once the diazeniumdiolated nitrilecompound is formed, the alkoxide or thiolate produces the correspondingimidate or thioimidate diazeniumdiolate.

Imidate and thioimidate diazeniumdiolates of formulae (I), (II), and(III) are useful as intermediate compounds in order to preparederivative diazeniumdiolate compounds with medicinal uses. Substitutednitric oxide-releasing thioimidates of formula (III) can be prepared bya method comprising (a) contacting a nitrile of the formula R¹CH₂CN witha metal alkoxide and an alcohol; (b) contacting the product of (a) withNO to form a diazeniumdiolated nitrile compound; (c) contacting thediazeniumdiolated nitrile compound with an alkoxide and alcohol (e.g.,NaOMe/HOMe); and (d) contacting the product of (c) with athiol-substituted compound to form a substituted nitric oxide-releasingthioimidate of formula (III). The thiol-substituted compound can be, forexample, L-cysteine, a protein, an enzyme, such as glutathione, or athiol-modified substrate. In a similar reaction, a primaryamine-substituted compound may be used in place of the thiol-substitutedcompound (e.g., L-lysine) in (d), in order to form an NO-releasingamidine.

In another aspect, the present invention provides novel nitricoxide-releasing amide compounds of formula (IV):

wherein R¹ is hydrogen, an unsubstituted or substituted C₁₋₁₂ straightchain alkyl, an unsubstituted or substituted C₃₋₁₂ branched chain alkyl,an unsubstituted or substituted C₃₋₁₂ straight chain alkenyl, anunsubstituted or substituted C₃₋₁₂ branched chain alkenyl, anunsubstituted or substituted alkoxy, nitrile, halo, an unsubstituted orsubstituted benzyl, an unsubstituted or substituted phenyl, anunsubstituted or substituted naphthyl, an unsubstituted or substitutedpiperazino, an unsubstituted or substituted morpholino, amino, anunsubstituted or substituted alkylamino, an unsubstituted or substitutedarylamino, an unsubstituted or substituted dialkylamino, anunsubstituted or substituted diarylamino, carboxyalkylamino,carboxydialkylamino, an unsubstituted or substituted tolyl, xylyl,anisyl, mesityl, an unsubstituted or substituted acetoxy, carboxy, anunsubstituted or substituted carboxyethyl, an unsubstituted orsubstituted alkylcarbonyl, thiol, an unsubstituted or substitutedalkylthio, an unsubstituted or substituted alkyloxy, carboxamido, anunsubstituted or substituted alkylcarboxamido, an unsubstituted orsubstituted dialkylcarboxamido, an unsubstituted or substituted phenoxy,an unsubstituted or substituted benzyloxy, phenylcarbonyl,benzylcarbonyl, an unsubstituted or substituted nitrophenyl,trialkylsilyl or nitro; and M^(+x) is a pharmaceutically acceptablecation, x is the valence of the cation, and a and b are the smallestintegers that result in a neutral compound. The substituent R¹ can befurther substituted with at least one nitric oxide-releasing functionalgroup.

The substituent R¹ of formula (IV) can be substituted with 1 to 3substituents that are independently selected from the group consistingof alkyl, aryl, such as phenyl or naphthyl, alkoxy, aryloxy, acyloxy,benzyl, benzyloxy, acetyl, carboxyl, carboxyalkyl, carboxyalkylamido,carboxydialkylamido, carboxamido, alkylcarbonyl, arylamino, diarylamino,nitrile, tolyl, xylyl, mesityl, anisyl, pyrrolidinyl, formyl, dioxane,alkylthiol, heteroaryl, such as pyran, pyrrole, furan, thiophene,thiazole, pyrazole, pyridine, or pyrimidine, phenylcarbonyl,benzylcarbonyl, nitrophenyl, trialkylsilyl, nitro, sulfonyl,nitrobenzyl, trialkylammonium, tetrahydrofuranyl, tetrahydropyranyl,piperdinyl, morpholinyl, halo, cyano, hydroxy, thiol, cycloalkyl, amino,alkylamino, and dialkylamino.

In addition, a bis-diazeniumdiolated amide compound of formula (IV) canbe prepared from an imidate diazeniumdiolate of formula (II).Substituted nitric oxide-releasing amides of formula (IV) can beprepared by a method comprising (a) contacting a nitrile of the formulaR¹CH₂CN with a metal alkoxide and an alcohol; (b) contacting the productof (a) with NO to form a diazeniumdiolated nitrile compound; (c)contacting the diazeniumdiolated nitrile compound with an alkoxide andalcohol (e.g., NaOMe/HOMe); and (d) contacting the product of (c) withhydroxide ion to form a substituted nitric oxide-releasing amide offormula (IV), wherein R¹ is discussed herein. Preferably the hydroxideion source is water, a Group I or II hydroxide (e.g., NaOH, KOH,Mg(OH)₂), or combinations thereof. Alternatively, thebis-diazeniumdiolated amide of formula (IV) can be prepared by directhydrolysis of the diazeniumdiolated nitrile compound. The methodcomprises (a) contacting a nitrile of the formula R¹CH₂CN with a metalalkoxide and an alcohol; (b) contacting the product of (a) with NO toform a diazeniumdiolated nitrile compound; (c) contacting thediazeniumdiolated nitrile compound with hydroxide ion to form asubstituted nitric oxide-releasing amide of formula (IV), wherein R¹ isdiscussed herein.

Any combination of metal alkoxide/alcohol or metal thiolate/thiol systemthat is capable of reaction with a nitrile to form an imidate orthioimidate when exposed to NO, results in the compounds, which arewithin the scope of the invention. Suitable metal alkoxides are of theformula MOR², wherein M and R² are as discussed above. Suitable alcoholsare of the formula HOR², wherein R² is as discussed above. Thesubstituent R² of the metal alkoxide and the alcohol can be the same ordifferent. Preferably, R² of the metal alkoxide and the alcohol are thesame. Similarly, suitable metal thiolates are of the formula MSR²,wherein M and R² are as discussed above. Suitable thiols are of theformula HSR², wherein R² is as discussed above. The substituent R² ofthe metal thiolate and the thiol can be the same or different.Preferably, R² of the metal thiolate and the thiol are the same. Alsoincluded in the invention are modifications to the above-describedsyntheses such as, for example, the use of a metal hydroxide incombination with an alcohol (e.g., MOH/HOR²). Combinations of metalhydroxides and metal alkoxides with an alcohol can also be used.

The nitriles of Equation 1 result in imidate and thioimidatediazeniumdiolates of formulae (I), (II), and (III) upon exposure tonitric oxide gas. To prepare the diazeniumdiolates of the presentinvention, nitrile, imidate, or thioimidate compounds are exposed tonitric oxide gas at a nitric oxide gas pressure as low as about 1 atm.Preferably, the pressure is at least about 1 atm, and more preferably,it is at least about 2 atm.

In another aspect, the invention provides compositions, includingpharmaceutical compositions, comprising the novel imidatediazeniumdiolates, the novel thioimidate diazeniumdiolates, the novelamide diazeniumdiolates or combinations thereof. Preferably, thepharmaceutical compositions further comprise a pharmaceuticallyacceptable carrier.

One skilled in the art will appreciate that suitable methods ofadministering a diazeniumdiolate composition of the present invention toan animal, e.g., a mammal such as a human, are known, and, although morethan one route can be used to administer a particular composition, aparticular route can provide a more immediate and more effectivereaction than another route. Pharmaceutically acceptable carriers arealso well known to those who are skilled in the art. The choice ofcarrier will be determined, in part, both by the particular compositionand by the particular method used to administer the composition.Accordingly, there is a wide variety of suitable formulations of thepharmaceutical compositions of the present invention.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the diazeniumdiolate dissolvedin diluents, such as water or saline, (b) capsules, sachets or tablets,each containing a predetermined amount of the active ingredient, assolids or granules, (c) suspensions in an appropriate liquid, and (d)suitable emulsions.

Tablet forms can include one or more of lactose, mannitol, cornstarch,potato starch, microcrystalline cellulose, acacia, gelatin, colloidalsilicon dioxide, croscarmellose sodium, talc, magnesium stearate,stearic acid, and other excipients, colorants, diluents, bufferingagents, moistening agents, preservatives, flavoring agents, andpharmacologically compatible carriers. Lozenge forms can comprise theactive ingredient in a flavor, usually sucrose and acacia or tragacanth,as well as pastilles comprising the active ingredient in an inert base,such as gelatin and glycerin or sucrose and acacia emulsions, gels, andthe like containing, in addition to the active ingredient, such carriersas are known in the art.

The diazeniumdiolates of the present invention, alone or in combinationwith other suitable components, can be made into aerosol formulations tobe administered via inhalation. These aerosol formulations can be placedinto pressurized acceptable propellants, such asdichlorodifluoromethane, propane, nitrogen, and the like.

Formulations suitable for parenteral administration include aqueous andnon-aqueous solutions, isotonic sterile injection solutions, which cancontain anti-oxidants, buffers, bacteriostats, and solutes that renderthe formulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The formulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid carrier, for example, water, for injections, immediatelyprior to use. Extemporaneous injection solutions and suspensions can beprepared from sterile powders, granules, and tablets of the kindpreviously described.

The dose administered to an animal, particularly a human, in the contextof the present invention should be sufficient to effect a therapeuticresponse in the animal over a reasonable time frame. The dose will bedetermined by the strength of the particular compositions employed(taking into consideration, at least, the rate of NO evolution, theextent of NO evolution, and the bioactivity of any decompositionproducts derived from the diazeniumdiolates) and the condition of theanimal (e.g., human), as well as the body weight of the animal (e.g.,human) to be treated. The size of the dose also will be determined bythe existence, nature, and extent of any adverse side effects that mightaccompany the administration of a particular composition. A suitabledosage for internal administration is 0.01 to 100 mg/kg per day. Apreferred dosage is 0.01 to 35 mg/kg per day. A more preferred dosage is0.05 to 5 mg/kg per day. A suitable concentration of thioimidate orimidate diazeniumdiolates in pharmaceutical compositions for topicaladministration is 0.05 to 15% (by weight). A preferred concentration isfrom 0.02 to 5%. A more preferred concentration is from 0.1 to 3%.

Nitric oxide-releasing imidates, thioimidates, and amides are useful forthe therapeutic treatment of many biological disorders. For example,thioimidate diazeniumdiolates are useful as fungicides (buthiobate) andin drug design for anticancer therapy. Reaction with amines will producenovel amidines of a class of compounds known for their antiviral,antibacterial, antifungal, and antiprotozoal properties.

The present invention also provides methods of using a nitricoxide-releasing diazeniumdiolate of formula (I), (II), (III) or (IV) ofthe invention or a substituted thioimidate diazeniumdiolate of thepresent invention. In one embodiment, a method of treating an animal,e.g., a mammal such as a human, with a biological disorder treatablewith nitric oxide, is provided. The method comprises administering tothe animal (e.g., human), in need thereof an amount of adiazeniumdiolate of formula (I), (II), (III) or (IV) of the invention, asubstituted thioimidate diazeniumdiolate of the present invention, orcompositions thereof sufficient to treat the biological disorder in theanimal (e.g., human). In this embodiment, “biological disorder” can beany biological disorder, including hypertension, sickle cell anemia,leishmania, restenosis, cancer, impotency, platelet aggregation, and abiological disorder due to a genetic defect or infection with aninfectious agent, such as a virus, bacterium, fungus or parasite, aslong as the disorder is treatable with nitric oxide.

The diazeniumdiolates of the invention are useful for treating ananimal, e.g., a mammal such as a human, for infection with, for example,a virus, a bacterium, or a parasite. The method comprises administeringto the animal, e.g., human, an amount of a diazeniumdiolate of formula(I), (II), (III) or (IV) of the invention, a substituted thioimidatediazeniumdiolate of the present invention, or compositions thereofsufficient to treat the infection in the animal.

The diazeniumdiolates of formula (I), (II), (III) or (IV) of theinvention, a substituted thioimidate diazeniumdiolate of the presentinvention, or compositions thereof are useful for treating an animal,e.g., a mammal such as a human, for cancer. The method comprisesadministering to the animal, e.g., human, an amount of adiazeniumdiolate of formulae of formula (I), (II), (III) or (IV) of theinvention, a substituted thioimidate diazeniumdiolate of the presentinvention, or compositions thereof sufficient to prevent the growth ormetastasis of the cancer in the animal (e.g., human) or to render itmore susceptible to radiation or chemotherapy.

The imidate and thioimidate diazeniumdiolates of the invention areuseful for treating an inanimate object for the presence of apotentially infectious virus, bacterium, or parasite. The methodcomprises contacting the inanimate object with an amount of andiazeniumdiolate of formula (I), (II), (III) or (IV) of the invention, asubstituted thioimidate diazeniumdiolate of the present invention, orcompositions thereof sufficient to reduce the presence of thepotentially infectious virus, bacterium or parasite. By “potentiallyinfectious” is meant the capability of infecting an animal, e.g., amammal such as a human.

The present invention provides a method of treating sickle cell anemiawith an imidate or thioimidate diazeniumdiolate of formula (I), (II) or(III) or compositions thereof. By way of example and not in limitationof the invention, the imidate diazeniumdiolate of1,4-phenylenediacetonitrile is useful in the treatment of sickle cellanemia. The compound 1,4-phenylenediacetonitrile is converted to thenitric oxide-releasing imidate by reaction with NO gas in alcoholicsodium methoxide as illustrated by Equation 2.

Compounds of the present invention and compositions thereof are usefulfor the treatment of leishmania. The present invention provides a methodof treating leishmania by administering an effective amount of acompound of formula (I), (II), (III) or (IV) of the invention, asubstituted thioimidate diazeniumdiolate of the present invention, orcompositions thereof. Preferably, the NO-releasing protein iscross-linked to the tubulin protein. The cross-linking molecule ispreferably chosen to disrupt tubulin polymerization and the release ofNO would augment the NO produced from the macrophages and impair theability of the parasite to form microtubules via nitration.

In another aspect of the invention, the metabolites of the organicmoiety of the diazeniumdiolate compounds of the present invention havemedicinal purposes as well. After the diazeniumdiolate compound offormula (I), (II), (III) or (IV) of the invention or substitutedthioimidate diazeniumdiolate of the present invention release NO underphysiological conditions, part of the organic moiety (e.g., the N₂O₂group) decomposes and the decomposition product, an oxime (hydroximinocompound) in many cases, has medicinal purposes (e.g., fungicidal). See,e.g., U.S. Pat. Nos. 5,264,484 and 4,874,786, S.U. Patent No. 1,094,273,J.P. Patent No. 05148104, D.E. Patent No. 2616089, and Massolini, etal., Farmaco, Ed. Sci., 42(2), 117–124 (1987), which are incorporated byreference. Therefore, the compounds of the present invention serve adual purpose: one in which the release of NO is beneficial and anotherin which the metabolite is medicinally useful.

Organic polymer beads with a free amino group are capable of reactingwith nitric oxide releasing imidates to form nitric oxide releasingpolymers. Organic polymer beads are currently used to immobilizeproteins to their surface via cross-linking reagents. Organic polymerbeads are ideal for reaction with the nitric oxide-releasing imidates togenerate polymers that can be a steady source of nitric oxide. Theconditions used to form the amidine linkage, typically pH of about8.0–9.0 and room temperature, are well suited to the imidatediazeniumdiolates of the invention because the diazeniumdiolate groupsare stable in that environment. Nitric oxide-releasing polymers haveextensive applications in the biomedical field as medical devices basedupon the novel properties of nitric oxide.

The nitric oxide-releasing functional group can also be that of apolymer, e.g., a nitric oxide-releasing imidate/thioimidate/amide boundto a polymer, similar to those described in, for example, U.S. Pat. Nos.5,405,919, 5,525,357, 5,632,981, 5,650,447, 5,676,963, 5,691,423, and5,718,892, and incorporated herein by reference. By “bound to a polymer”it is meant that the nitric oxide-releasing imidate/thioimidate/amide,such as those described by formulae (I)–(IV) is associated with, partof, incorporated with, or contained within the polymer matrix physicallyor chemically. Physical association or bonding of the nitricoxide-releasing imidate/thioimidate/amide to the polymer may be achievedby co-precipitation of the polymer with the nitric oxide-releasingimidate/thioimidate/amide as well as by covalent bonding of the complexto the polymer. Chemical bonding of the nitric oxide-releasingimidate/thioimidate/amide to the polymer may be by, for example,covalent bonding of the imidate/thioimidate moiety of the nitricoxide-releasing imidate/thioimidate/amide to the polymer such that theimidate/thioimidate/amide residue to which the NONO group is attachedforms part of the polymer itself, i.e., is in the polymer backbone, oris attached to a group or groups pendant to the polymer backbone. If thediazeniumdiolates of the present invention are chemically bound to thepolymer/biopolymer, then the diazeniumdiolates are bound to thepolymer/biopolymer by at least one functional group on the polymer orbiopolymer. Preferably, more than one diazeniumdiolate compound of thepresent invention is chemically bound per molecule of thepolymer/biopolymer. The manner in which the nitric oxide-releasingimidate/thioimidate/amide is associated, part of, or incorporated withor contained within, i.e., “bound” to the polymer, is inconsequential tothe invention and all means and degrees of association, incorporation orbonding are contemplated herein.

Any of a wide variety of polymers can be used in the context of theinvention. Illustrative of polymers suitable for use in the inventionare polyolefins, such as polystyrene, polypropylene, polyethylene,polytetrafluorethylene, polyvinyl chloride, polyvinylidene difluoride,and polyethers such as polyethylene glycol, polysaccharides such asdextran, polyesters such as poly(lactide/glycolide), polyamides such asnylon, polyurethanes, polyethylenimine, biopolymers such as peptides,polypeptides, enzymes, polysaccharides, proteins, oligonucleotides,antibodies and nucleic acids, starburst dendrimers, and the like. Forexample, chitosan is a polysaccharide containing numerous primary aminegroups. The term “biopolymers”, as used herein, also include monomericunits of larger biopolymers such as monosaccharides, amino acids ornucleotides.

The advantage of preparing diazeniumdiolate compounds bound to a(bio)polymer by the methods of the present invention is that thediazeniumdiolate compound is formed first and then bound to the polymer.This methods enables a whole new class of compounds to be preparedbecause the (bio)polymer is not directly exposed to the potentiallydeleterious effects of nitric oxide gas or any nitrogen oxide that mightform therefrom in the presence of oxygen.

The imidate diazeniumdiolate compounds of formula (II) also serve ascross-linking agents. Homobifunctional imidates are known in the art toserve as cross-linkers, particularly in the cross-linking of proteins.See, e.g., Ahmadi, et al. FEBS Lett. 94(2), 365–367 (1978); Brandon,Cellular and Molecular Biology 26, 569–573 (1980), Brew, et al., J.Biol. Chem., 250(4) 1434–1444 (1975). Bifunctional imidatediazeniumdiolates are within the scope of the present invention, suchas, in non-limiting examples, where R is a nitrile or alkyl groupsubstituted with a nitrile. The methods of preparing such bifunctionalimidate diazeniumdiolates are the same as those for preparing compoundsof formula (II), which are described herein. The bifunctional imidatediazeniumdiolate can cross-link any suitable compound, such as a polymeror biopolymer described herein. The cross-linking can occur eitherbetween more than one compound (e.g., two polymer chains) or betweensites within the same molecule.

Another aspect of this invention includes a method for preparing anitric oxide-releasing imidate/thioimidate/amide that is capable ofbeing bound to a substrate, where the method includes contacting thediazeniumdiolate with a substrate. Preferably the substrate has moietiesthat allow for chemical bonding of the NO-releasingimidate/thioimidate/amide to the substrate. See, for example, U.S.patent application Ser. No. 2001/0003599, which is incorporated hereinin its entirety. In a specific example, a stainless steel coupon can betreated such that free thiol groups reside on the surface. AnNO-releasing imidate of formula (II) can be reacted with the stainlesssteel coupon to form a substrate coated with an NO-releasing substitutedthioimidate.

The substrate can be of any suitable biocompatible material, such asmetal, glass, ceramic, or plastic or rubber. Preferably, the substrateis metal. The substrate used in the preparation of the medical devicecan be derived from any suitable form of a biocompatible material, suchas, for example, a sheet, a fiber, a tube, a fabric, an amorphous solid,an aggregate, dust or the like.

Metal substrates suitable for use in the invention include, for example,stainless steel, nickel, titanium, tantalum, aluminum, copper, gold,silver, platinum, zinc, silicon, magnesium, tin, alloys, coatingscontaining any of the above and combinations of any of the above. Alsoincluded are such metal substrates as galvanized steel, hot dippedgalvanized steel, electrogalvanized steel, annealed hot dippedgalvanized steel and the like. Preferably, the metal substrate isstainless steel.

Glass substrates suitable for use in the invention include, for example,soda lime glass, strontium glass, borosilicate glass, barium glass,glass-ceramics containing lanthanum as well as combinations thereof.

Ceramic substrates suitable for use in the invention include, forexample, boron nitrides, silicon nitrides, aluminas, silicas,combinations thereof, and the like.

Plastic substrates suitable for use in the invention include, forexample, acrylics, acrylonitrile-butadiene-styrene, acetals,polyphenylene oxides, polyimides, polystyrene, polypropylene,polyethylene, polytetrafluoroethylene, polyvinylidene, polyethylenimine,polyesters, polyethers, polylactones, polyurethanes, polycarbonates,polyethylene terephthalate, as well as copolymers and combinationsthereof. Typical rubber substrates suitable for use in the inventioninclude, for example, silicones, fluorosilicones, nitrile rubbers,silicone rubbers, fluorosilicone rubbers, polyisoprenes, sulfur-curedrubbers, isoprene-acrylonitrile rubbers, and the like. Silicones,fluorosilicones, polyurethanes, polycarbonates, polylactones, andmixtures or copolymers thereof are preferred plastic or rubbersubstrates because of their proven bio- and hemocompatability when indirect contact with tissue, blood, blood components, or bodily fluids.

Other suitable substrates include those described in WO 00/63462, andincorporated herein by reference.

The invention provides medical devices which are capable of releasingnitric oxide when in use, but which are otherwise inert to nitric oxiderelease. In particular, NO-releasing functional groups are bound to asubstrate that is coated with an imidate or thioimidatediazeniumdiolate. The term “bound” as used herein includes covalentbonds, ionic bonds, van der Waal forces, hydrogen bonding, electrostaticbonding, and all other methods for attaching organic chemical functionalgroups to a substrate.

A “medical device” refers to any device having surfaces that contacttissue, blood, or other bodily fluids in the course of their use oroperation, which are found in or are subsequently used in patients oranimals. Medical devices include, for example, extracorporeal devicesfor use in surgery, such as blood oxygenators, blood pumps, bloodstorage bags, blood collection tubes, blood filters including filtrationmedia, tubing used to carry blood and the like which contact blood whichis then returned to the patient or animal. Medical devices also includeendoprostheses implanted in a human or animal body, such as vasculargrafts, stents, pacemaker leads, heart valves, and the like, that areimplanted in blood vessels or the heart. Medical devices also includedevices for temporary intravascular use such as catheters, guide wires,amniocentesis and biopsy needles, cannulae, drainage tubes, shunts,sensors, transducers, probes and the like which are placed into theblood vessels, the heart, organs or tissues for purposes of monitoringor repair or treatment. Medical devices also include prostheses such asartificial joints such as hips or knees as well as artificial hearts.

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

The nitrites and all solvents were purchased from Aldrich ChemicalCompany (Milwaukee, Wis.). Nitric oxide was obtained form Matheson GasProducts (Montgomeryville, Pa.). The ¹H NMR and ¹³C NMR spectra wererecorded in D₂O (¹H at 300 MHz; ¹³C at 75 MHz). Ultraviolet spectra (10mM NaOH unless otherwise specified) and kinetic information wereobtained on a Hewlett Packard 8452A UV-visible spectrophotometer with athermostated cell holder. Infrared data was recorded on a Perkin-Elmer1430 infrared spectrometer. Nitric oxide was measured with a ThermalEnergy Analyzer model 502A (Thermedics, Inc., Woburn, Mass.). Meltingpoints are uncorrected. Elemental analyses were performed by AtlanticMicrolab, Inc. (Norcross, Ga.).

EXAMPLE 1

This Example describes a generalized procedure for the preparation ofnitric oxide (NO) and or nitroxyl (NO⁻) releasing imidates fromnitrites.

The appropriate nitrile was dissolved in methanol and 2 equivalents ofmetal alkoxide (25% sodium methoxide solution unless otherwise noted)were added. The solution was placed in a standard high pressure Parr®bottle. Argon was passed through the solution for 10 minutes beforeattachment to the NO apparatus. A complete description of these pressurereactions has been given in Hrabie et al. (J. Org. Chem. 58: 1472–1476(1993)). The Parr® bottle was then alternately pressurized to 1.4 atm(20 psi) with high purity argon and vented for a total of ten cycles.The vessel was then pressurized to 2.7 atm (40 psi) with NO gas whilestirring at room temperature. Additional NO was added as needed. Whenthe consumption of NO was complete, usually within about 24 hours,excess NO gas was vented, and the system was purged with argon. Theproduct was isolated by filtration, washed with diethyl ether, and driedin vacuo. A crystalline material can be obtained by dissolution of thepowder in a minimum amount of water and addition of a methanol-ethermixture until turbid. Crystallization occurs at 4° C. overnight.

EXAMPLE 2

This Example describes the preparation of benzyl diazeniumdiolatemethoxy imidate.

Benzyl cyanide (5 mL, 0.043 mol) was dissolved in 50 mL methanol in apressure bottle, and 2 equivalents of sodium methoxide (19.6 mL, 0.086mol) were slowly added. The solution was reacted with NO gas for 24hours and the product was isolated as a hydrate. Yield 5.25 g (35%); UVλ_(max)=264 nm (ε=14.0 mM⁻¹ cm⁻¹); Mp chars above 250° C.; ¹H NMR (300MHz, D₂O) 3.8 ppm (s, 3H), 7.2–7.6 ppm (m, 5H). Anal. Calcd forC₉H₉N₅O₅x2H₂O: C, 30.95; H, 3.75; N, 20.05. Found: C, 30.65; H, 3.85; N,19.79.

EXAMPLE 3

This Example describes the preparation of benzyl bis diazeniumdiolateethoxy imidate.

Benzyl cyanide (3 mL, 0.025 moles) was dissolved in 30 mL of ethanol,and 2 equivalents of potassium ethoxide (4.30 g, 0.051 moles) wereadded. The reaction was run as described in Example 1. A light tanproduct was obtained. Yield 4.48 g, 50%; Mp 150° C. dec.; UV λ_(max) (ε)262 nm (13.6 mM⁻¹ cm⁻¹); ¹H NMR (300 MHz, D₂O): 1.27 (t, 3H), 4.26 (q,2H), 7.47 ppm (s, 5H). Anal. Calcd for C₁₀H₁₁N₅K₂O₅: C, 33.42; H, 3.08;N, 19.49. Found: C, 33.48; H, 3.08; N, 19.17.

EXAMPLE 4

This Example describes the preparation of a derivative of benzyldiazeniumdiolate methoxy imidate with an electron donating substituenton the ring.

4-Methoxybenzyl cyanide (5 mL, 0.036 moles) was mixed with 50 mLmethanol, and 25% sodium methoxide (16.8 mL, 0.072 moles) was added. TheNO reaction was conducted as described in Example 1, and a white powderwas obtained as a hydrate. Yield 1.57 g, 11%; Mp 140° C. dec.; UVλ_(max) (ε) 262 nm (14.6 mM⁻¹ cm⁻¹); ¹³C NMR (75 MHz, D₂O): 57.34,58.28, 99.15, 116.67 (2C), 127.38, 133.14 (2C), 162.71, 168.13 ppm. ¹HNMR (300 MHz, D₂O): 3.85 (s, 3H), 3.87 (s, 3H), 7.01–7.40 ppm (AA′BB′4H). Anal. Calcd for C₁₀H₁₇N₅Na₂O₉: C, 30.23; H, 4.31; N, 17.63. Found:C, 30.44; H, 4.22; N, 17.31.

EXAMPLE 5

This Example describes the preparation of a derivative of benzyl bisdiazeniumdiolate methoxy imidate with an electron withdrawingsubstituent on the ring.

A solution of 4-chlorobenzyl cyanide (5 mL, 0.042 moles) was mixed with50 mL of methanol, and 25% sodium methoxide (20 mL, 0.084 moles) wasadded. The solution was allowed to react with 2.7 atm (40 psi) of NO gasovernight. Yield 6.92 g, 43%; UV λ_(max) (ε) 262 nm (14.5 mM⁻¹ cm⁻¹);¹³C NMR (75 MHz, D₂O): 57.44, 98.73, 131.30 (2C), 133.09(2C), 133.94,138.16, 167.69 ppm. ¹H NMR (300 MHz, D₂O): 3.83 (s, 3H), 7.40–7.49 (m,4H). Anal. Calcd for C₉H₁₀ClN₅Na₂O₆: C, 29.56; H, 2.76; N, 19.15. Found:C, 29.42; H, 2.92; N, 18.89.

EXAMPLE 6

This Example describes the preparation of benzyl diazeniumdiolatenitrile and its subsequent conversion to an imidate.

Benzyl cyanide (3 mL, 0.025 moles) was mixed with 25 mL THF and 10 mL ofa 1.0 M solution of trimethylsilanoate sodium salt (10 mL, 0.01 moles)was added. After reaction with NO gas for 24 hours, the precipitate wasfiltered, washed, and dried. Yield 1.6 g, 100%; mp=192–195° C.; UVλ_(max) (ε) 266 nm (12.5 mM⁻¹ cm⁻¹); IR; (KBr pellet) 2240 cm⁻¹; ¹³C NMR(75 MHz, D₂O): 94.08, 116.86, 130.53 (2C), 132.03 (2C), 133.25, 134.07(2C) ppm. ¹H NMR (300 MHz, D₂O): 7.58 (s, 5H).

A sample of the bis diazeniumdiolated product thus obtained (100 mg) wasthen dissolved in a minimum of water (0.5 mL) and diluted with methanol(5 mL). Then 5 drops of 25% sodium methoxide were added and the solutionwas allowed to stir at room temperature overnight. Diethyl ether wasadded until the solution was turbid at which point it was allowed toprecipitate in the refrigerator at 4° C. The product, identified by NMRand IR spectra, was identical to the diazeniumdiolated methoxy imidateproduced in Example 2.

EXAMPLE 7

This Example describes the conversion of benzyl diazeniumdiolate ethoxyimidate to benzyl diazeniumdiolate methoxy imidate.

Benzyl diazeniumdiolate ethoxy imidate was synthesized as described inExample 3, dissolved in a minimum amount of water (0.5 mL), and dilutedwith methanol (5.0 mL). The solution was stirred at room temperatureovernight. Diethyl ether was used to precipitate out the benzyldiazeniumdiolated methoxy imidate overnight at 4° C. Conversion wasquantitative.

EXAMPLE 8

This Example describes the conversion of 3,4,5-trimethoxy benzyldiazeniumdiolate methoxy imidate to 3,4,5-trimethoxy benzyldiazeniumdiolate amide.

A solution of 3,4,5-trimethoxybenzyl cyanide (2.00 g, 0.0096 moles) in10 mL methanol was treated with 25% sodium methoxide (4.41 mL, 0.019moles). The solution was degassed and exposed to 2.7 atm (40 psi) of NOgas for 6 hours. After filtration, the crude material was dissolved inwater (2 mL), which converted the imidate to the amide in situovernight. Yield 1.06 g, 26%; mp=187–190° C.; UV λ_(max) (ε) 258 nm(broad) (11.0 mM⁻¹ cm⁻¹); ¹³C NMR (75 MHz D₂O): 57.35, 58.97, 59.11,63.74, 93.75, 98.85, 108.43, 109.41, 116.67, 129.46, 131.65, 140.50,154.85, 155.44, 167.79 ppm. ¹H NMR (300 MHz D₂O): 3.85 (s, 3H), 3.89 (s,6H), 6.93 (s, 2H). Anal. Calcd for C₁₁H₁₇N₅Na₂O₁₀: C, 31.07; H, 4.03; N,16.47. Found: C, 31.37; H, 4.05; N, 16.29.

EXAMPLE 9

This Example describes the conversion of 1-naphthyl diazeniumdiolateimidate to 1-naphthyl diazeniumdiolate amide.

1-Naphthylacetonitrile (2.36 g, 0.014 moles) was dissolved in 25 mL ofmethanol, and 25% sodium methoxide (6.45 mL, 0.028 moles) was added. Thesolution was allowed to react with NO gas at 2.7 atm (40 psi) overnightand was then filtered. The product was dissolved in water andprecipitated with methanol/ether in the refrigerator. Yield 1.07 g, 20%;mp 130° C. dec.; UV λ_(max) (ε) 274 nm (17.1 mM⁻¹ cm⁻¹); ¹³C NMR (75MHz, D₂O): 94.31, 116.21, 125.88, 127.82, 128.53, 129.32, 129.45,130.62, 131.83, 132.31, 135.28, 136.50 ppm. ¹H NMR (300 MHz, D₂O):7.39–8.14 ppm (m, 7.5H). Anal. Calcd for C₁₂H₁₃N₅Na₂O₇: C, 37.41; H,3.40; N, 18.18; Na, 11.94. Found: C, 37.92; H, 3.50; N, 17.95; Na,11.82.

EXAMPLE 10

This Example describes the reaction between an NO releasing imidate andan amino acid to form an NO-releasing substituted thioimidate.

The benzyl diazeniumdiolated imidate (1.46 g, 0.0042 moles) wasdissolved in 3 mL 0.01 mM NaOH and mixed with a solution of L-cysteine(5.0 g in 20 mL 0.01 mM NaOH) at room temperature. The solution turnedlight pink, and a precipitate appeared after several minutes. After 24hours, the solution was filtered, washed with ether, and dried in vacuo.Yield: 1.78 g. NO release in pH 7.4 buffer at 37° C. from the productwas measured qualitatively.

EXAMPLE 11

This Example illustrates the production of NO and N₂O bydiazeniumdiolated imidates.

For the detection of NO, selected compounds from the examples weredissolved in either pH 7.4 buffer or 10% H₂SO₄, and the headspace wasswept with argon into a chemiluminescence detector. The detection ofnitrous oxide (N₂O), formed from the dimerization and dehydration ofHNO, was done by gas chromatography in both pH 7.4 buffer and 0.1 M HCl.The results are shown in Table 1.

Compound NO N₂O from Example Solution (mole gas/mole cmpd) (molegas/mole cmpd 2 pH 7.4 0.36 — 2 acid 0.46 1.52 4 pH 7.4 0.34 1.08 4 acid0.19 1.07 5 pH 7.4 0.38 1.06 9 pH 7.4 0.16 — 9 acid 0.02 —

EXAMPLE 12

This Example illustrates the measurement of the rate of NO releasingdecay of diazeniumdiolate imidates in solution.

A stock solution of the NO releasing imidate in 10 mM NaOH was prepared.A quartz cuvette was filled with 3 mL of pH 7.4 phosphate buffer andfitted to a thermostated cell holder in the UV-visiblespectrophotometer. A stirring bar was added to the cell to providesample homogeneity. After 5 minutes of equilibration to 25° C., analiquot of the stock solution (10 μl) was injected into the cuvette toinitiate the kinetic experiment. The NO releasing imidates generallyshow good first order decay kinetics. The half-life data for selectedcompounds is shown in Table 2.

Compound from t_(1/2) Example (seconds) 2 738 4 113 5 198

EXAMPLE 13

This Example describes the conversion of acetonitrile to an NO-releasingimidate.

A solution of 100 mL acetonitrile and 10 mL (0.043 moles) 25% sodiummethoxide is degassed and placed under 2.7 atm (40 psi) of NO gas. Anoff-white product is obtained overnight, which is worked up in theabove-described manner to produce a product that was explosive uponheating. Brown gas is evolved upon acidification and NO is detected bychemiluminescence. Yield: 3.1 g; mp detonates at 182° C.; UV-visλ_(max)=264 nm in 10 mM NaOH.

EXAMPLE 14

This Example describes the conversion of methylaminoacetonitrile to anNO-releasing imidate.

A solution of 3.8 g (0.035 moles) methylaminoacetonitrile hydrochloridewas first converted to the free base with 25% sodium methoxide. Thesodium chloride was filtered and the filtrate diluted with 50 mL ofmethanol. To this solution, 16 mL (0.07 moles) of 25% sodium methoxidewas added, and the solution was degassed and pressurized with NO.Precipitate is noticeable within an hour but consumption of NO gas issluggish. The white product obtained by filtration produces gas bubblesupon acidification. Yield: 0.856 g; UV-vis λ_(max)=262 nm in 10 mM NaOH.

EXAMPLE 15

This Example describes the conversion of propionitrile to anNO-releasing imidate.

A solution of 25 mL propionitrile and 12 mL (0.052 moles) sodiummethoxide was degassed and pressurized to 2.7 atm (40 psi) of NO gas.Within 2 hours precipitate was observed and a white powder was obtainedby filtration. Yield: 670 mg; UV-vis λ_(max)=262 nm in 10 mM NaOH.

EXAMPLE 16

This Example describes the conversion of butyronitrile to anNO-releasing imidate.

A solution of 50 mL diethyl ether, 1 mL methanol, and 25 mLbutyronitrile was mixed with 10 mL (0.043 moles) of 25% sodiummethoxide, degassed, and placed under 2.7 atm (40 psi) NO gas. A grayishpowder was obtained by filtration. Yield: 1.07 g; UV-vis λ_(max)=262 nmin 10 mM NaOH.

EXAMPLE 17

This Example describes preparing the imidate first and then forming thediazeniumdiolated imidate in situ.

A solution of 5 mL (0.079 moles) chloroacetonitrile was allowed to reactin 10 mL of a methanol/methoxide solution (0.043 moles methoxide) atroom temperature for 35 minutes. The solution was degassed and placedunder 2.04 atm (30 psi) NO gas. The tan colored precipitate was isolated2 days later by filtration and dried for a yield of 2.35 grams. Thematerial turns blue in a 2.0 M solution of HCl with effervescence andhas a UV-vis λ_(max)=262 nm in water.

EXAMPLE 18

This Example describes preparing the bis-diazeniumdiolated amidedirectly from the diazeniumdiolated nitrile in situ.

This method precludes the formation of an imidate by avoiding the sodiummethoxide/methanol method. Sodium hydroxide (1.73 g, 0.043 moles) wasadded to a stirred solution of 2.5 mL (0.0216 moles) of benzyl cyanide,10 mL diethyl ether and 1 mL of water. The solution was degassed andpressurized to 2.7 atm (40 psi) of NO gas. The solution darkened as thereaction proceeded and a product precipitated overnight. The product wasisolated by filtration and dried. There was no nitrile absorption in theIR spectrum. Yield: 726 mg; UV-vis λ_(max)=262 nm in 10 mM NaOH.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. Recitation of ranges of values herein are merely intended toserve as a shorthand method of referring individually to each separatevalue falling within the range, unless otherwise indicated herein, andeach separate value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein, isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention unless otherwise claimed. Nolanguage in the specification should be construed as indicating anynon-claimed element as essential to the practice of the invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention. Ofcourse, variations of those preferred embodiments will become apparentto those of ordinary skill in the art upon reading the foregoingdescription. The inventors expect skilled artisans to employ suchvariations as appropriate, and the inventors intend for the invention tobe practiced otherwise than as specifically described herein.Accordingly, this invention includes all modifications and equivalentsof the subject matter recited in the claims appended hereto as permittedby applicable law. Moreover, any combination of the above-describedelements in all possible variations thereof is encompassed by theinvention unless otherwise indicated herein or otherwise clearlycontradicted by context.

1. A compound of the formula (I);

wherein X is O or S; R¹ is hydrogen, an unsubstituted or substitutedC₁₋₁₂ straight chain alkyl, an unsubstituted or substituted C₃₋₁₂branched chain alkyl, an unsubstituted or substituted C₃₋₁₂ straightchain alkenyl, an unsubstituted or substituted C₃₋₁₂ branched chainalkenyl, an unsubstituted or substituted alkoxy, halo, an unsubstitutedor substituted benzyl, an unsubstituted or substituted phenyl, amino, anunsubstituted or substituted alkylamino, an unsubstituted or substitutedarylamino, an unsubstituted or substituted dialkylamino, anunsubstituted or substituted diarylamino, an unsubstituted orsubstituted tolyl, xylyl, anisyl, mesityl, an unsubstituted orsubstituted alkylthio, an unsubstituted or substituted alkyloxy, anunsubstituted or substituted phenoxy, an unsubstituted or substitutedbenzyloxy, or nitro; R² is an unsubstituted or substituted C₁₋₁₂straight chain alkyl or an unsubstituted, substituted C₃₋₁₂ branchedchain alkyl, or a phenyl; R³ is hydrogen, an unsubstituted orsubstituted C₁₋₁₂ straight chain alkyl, an unsubstituted or substitutedC₃₋₁₂ branched chain alkyl, an unsubstituted or substituted C₃₋₁₂straight chain alkenyl, an unsubstituted or substituted C₃₋₁₂ branchedchain alkenyl, an unsubstituted or substituted C₃₋₈ cycloalkyl, benzylor substituted benzyl, or phenyl or substituted phenyl; and M^(+X) is apharmaceutically acceptable cation, x is the valence of the cation, anda and b are the smallest integers that result in a neutral compound,wherein the compound is bound to a polymer.
 2. The compound of claim 1,wherein the polymer is a biopolymer.
 3. The compound of claim 1, whereinR¹ is substituted with at least one nitric oxide-releasing functionalgroup.
 4. The compound of claim 1, wherein R¹ is unsubstituted orsubstituted benzyl or unsubstituted or substituted phenyl.
 5. Thecompound of claim 1, wherein R³ is hydrogen.
 6. The compound of claim 1,wherein each of R¹, R², and R³ is optionally substituted each with 1 to3 substituents independently selected from the group consisting ofalkyl, aryl, alkoxy, aryloxy, acyloxy, benzyl, benzyloxy, acetyl,carboxyl, carboxyalkyl, carboxyalkylamido, carboxydialkylamido,carboxamido, alkylcarbonyl, arylamino, diarylamino, nitrile, tolyl,xylyl, mesityl, anisyl, pyrrolidinyl, formyl, dioxane, alkylthiol,heteroaryl, such as pyran, pyrrole, furan, thiophene, thiazole,pyrazole, pyridine, or pyrimidine, phenylcarbonyl, benzylcarbonyl,nitrophenyl, trialkylsilyl, nitro, sulfonyl, nitrobenzyl,trialkylammonium, tetrahydrofuranyl, tetrahydropyranyl, piperdinyl,morpholinyl, halo, cyano, hydroxy, thiol, cycloalkyl, amino, alkylamino,and dialkylamino.
 7. A pharmaceutical composition comprising at leastone compound of claim 1 and a pharmaceutically acceptable carrier.
 8. Acompound of the formula (I):

wherein X is O or S; R¹ is hydrogen, an unsubstituted or substitutedC¹⁻¹² straight chain alkyl, an unsubstituted or substituted C³⁻¹²branched chain alkyl, an unsubstituted or substituted C₃₋₁₂ straightchain alkenyl, an unsubstituted or substituted C₃₋₁₂ branched chainalkenyl, an unsubstituted or substituted alkoxy, halo, an unsubstitutedor substituted benzyl, an unsubstituted or substituted phenyl, amino, anunsubstituted or substituted alkylamino, an unsubstituted or substitutedarylamino, an unsubstituted or substituted dialkylamino, anunsubstituted or substituted diarylamino, an unsubstituted orsubstituted tolyl, xylyl, anisyl, mesityl, an unsubstituted orsubstituted alkylthio, an unsubstituted or substituted alkyloxy, anunsubstituted or substituted phenoxy, an unsubstituted or substitutedbenzyloxy, or nitro; R² is an unsubstituted or substituted C₁₋₁₂straight chain alkyl or an unsubstituted, substituted C₃₋₁₂ branchedchain alkyl, or a phenyl; R³ is hydrogen, an unsubstituted orsubstituted C₁₋₁₂ straight chain alkyl, an unsubstituted or substitutedC₁₋₁₂ branched chain alkyl, an unsubstituted or substituted C₃₋₁₂straight chain alkenyl, an unsubstituted or substituted C₃₋₁₂ branchedchain alkenyl, an unsubstituted or substituted C₃₋₈ cycloalkyl, benzylor substituted benzyl, or phenyl or substituted phenyl; and M^(+X) is apharmaceutically acceptable cation, x is the valence of the cation, anda and b are the smallest integers that result in a neutral compound,wherein the compound is bound to a substrate.
 9. The compound of claim8, wherein R¹ is substituted with at least one nitric oxide-releasingfunctional group.
 10. The compound of claim 8, wherein R¹ isunsubstituted or substituted benzyl or unsubstituted or substitutedphenyl.
 11. The compound of claim 8, wherein R³ is hydrogen.
 12. Thecompound of claim 8, wherein each of R¹, R², and R³ is optionallysubstituted each with 1 to 3 substituents independently selected fromthe group consisting of alkyl, aryl, alkoxy, aryloxy, acyloxy, benzyl,benzyloxy, acetyl, carboxyl, carboxyalkyl, carboxyalkylamido,carboxydialkylamido, carboxamido, alkylcarbonyl, arylamino, diarylamino,nitrile, tolyl, xylyl, mesityl, anisyl, pyrrolidinyl, formyl, dioxane,alkylthiol, heteroaryl, such as pyran, pyrrole, furan, thiophene,thiazole, pyrazole, pyridine, or pyrimidine, phenylcarbonyl,benzylcarbonyl, nitrophenyl, trialkylsilyl, nitro, sulfonyl,nitrobenzyl, trialkylammonium, tetrahydrofuranyl, tetrahydropyranyl,piperdinyl, morpholinyl, halo, cyano, hydroxy, thiol, cycloalkyl, amino,alkylamino, and dialkylamino.
 13. The compound of claim 1, wherein thepolymer comprises polyolefin, polyether, polysaccharide, polyester,polyamide, polyurethane, or polyethylenimine.
 14. The compound of claim13, wherein the polymer comprises polystyrene, polypropylene,polyethylene, polytetrafluorethylene, polyvinyl chloride, polyvinylidenedifluoride, polyethylene glycol, dextran, chitosan,poly(lactide/glycolide), or nylon.
 15. The compound of claim 2, whereinthe biopolymer comprises a peptide, a polypeptide, an enzyme, a protein,an oligonucleotide, an antibody, or a nucleic acid.
 16. The compound ofclaim 8, wherein the substrate comprises metal, glass, ceramic, plastic,or rubber.
 17. The compound of claim 8, wherein the substrate is amedical device.
 18. The compound of claim 17, wherein the medical deviceis a blood oxygenator, a blood pump, a blood storage bag, a bloodcollection tube, a blood filter, tubing, a vascular graft, a stent, apacemaker lead, a heart valve, a catheter, a guide wire, anamniocentesis or biopsy needle, a cannula, a drainage tube, a shunt, asensor, a transducer, a probe, an artificial joint, or an artificialheart.